2
Q. Zhao et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
O
OH
3 2
CH N(CH )
HO
O
N
N
HO
O
N
N
O
O
O
2
N
N
O
O
irinotecan
topotecan
Figure 1. Structure of irinotecan and topotecan.
(
IC50) values, which are the concentrations leading to 50% cell
O
O
death in vitro, were obtained. In addition, nineteen compounds
with the best anti-proliferative activity were tested at 50
their capability to inhibit Top I-mediated DNA relaxation.
O
lM for
O
O
N
Table 1 presents cytotoxic activities of indenoisoquinoline
derivatives and manifests that substitution pattern at the inde-
noisoquinoline 3-position and the lactam side chain has a signifi-
cant effect on the in vitro cytotoxic activities of the molecules.
From the listed data, we can conclude that the biological activity
is greatly improved with the utilization of the nitro, amino, aceta-
mido and methoxyl groups at 3-position of indenoisoquinolines
and that follows this trends: amino and acetamido > methoxyl >
nitro. Obviously, the effects of electron-donate groups on enhanc-
ing the biological activity of indenoisoquinoline Top I inhibitors are
better than electron-withdrawing groups. Comparing series A, B, C
and D compounds, we can see that series B, C and D compounds
had nearly 2–100-fold preferable cytotoxicity than series A com-
O
NSC 314622
Figure 2. Structure of NSC 314622.
substituent (series B). In order to probe the influence of electronic
effect to the isoquinoline ring, we also synthesized two series com-
pounds with an acetamido or a methoxyl group instead of the nitro
group (series C and D).
The synthesis of the four series of indenoisoquinoline deriva-
tives were demonstrated in Schemes 1 and 2. 2-Chlorobenzoic acid
was nitrated, substituted, hydrolyzed, decarboxylated and cyclized
to provide compound 5. Condensation of compound 5 with Schiff
base (19) yielded carboxylic acid 6. Compound 6 was cyclized to
provide 7, which was treated with the corresponding amines to
give the series A compounds (8a–8e). Compounds 8a–8e were
hydrogenated to give the series B compounds (9a–9e). Compounds
pounds (nitro group) with an IC50 of 0.048–29.91 lM range. Series
B and C compounds with amino and acetamido groups at 3-posi-
tion was the most potent compounds we synthesized. Comparing
the different substituents appended to the lactam side chain, we
can also conclude that the activity of nitrogenous substituted com-
pounds was significantly better than the chloro ones. For instant,
compounds 10a–e and 18a–e were more potent than compounds
9
a–9e were acetylated to yield the series C compounds (10a–10e).
Compound 4 was hydrogenated, substituted, esterified, substi-
tuted, hydrolyzed and cyclized to provide compound 16. Conden-
sation of compound 16 with Schiff base (19) yielded carboxylic
acid 17. Carboxylic acid 17 was cyclized to yield compound 18,
and then treated with appropriate amines to give the series D com-
pounds (18a–18e). Synthesis and chemical data (including
NMR, MS) in details were provided in supporting information.
The chemical data including H NMR, C NMR, MS, IR, and elemen-
tal analysis of compound 9a was provided in Ref. 22
To explore the in vitro anti-proliferative activity of these inde-
noisoquinoline derivatives, we used the 1-N-methyl-5-thiotetra-
zole (MTT)-based test in there human cancer cell lines HepG2
1
0 and 18 in cell killing activity. Therefore, introducing nitroge-
nous group at lactam side chain resulted in a rapid increase in bio-
logical activity. The data manifests that the properties of the
substituents at lactam side chain is a crucial factor for the potency
and that follows this trends: 4-methyl piperazinyl > pyrrolidyl,
piperidyl, diethylamino ꢀ morpholinyl. Compounds 9a and 10a
were discovered to be the best of all compounds we synthesized,
1
H
1
13
with IC50 values of 0.08, 0.12
lM (HepG2), 0.048, 0.062 lM
(
A549) and 0.117, 0.233 M (HCT-116), respectively; approxi-
l
mately 2–100-fold more potent than HCPT and TPT in this biolog-
ical assay.
Figure 4 shows the effects of indenoisoquinoline derivatives on
Top I-mediated DNA relaxation activity. We compared the inhibi-
tory activities of these compounds with a famous DNA-Top I inhi-
bitor, CPT. From the picture, we can see that all the tested
compounds have moderate inhibitory activities on Top I which
was right to the anti-proliferative activities in MTT assay. Com-
pounds 9a and 10a show best activity, which is as potent as CPT.
In summary, 22 novel indenoisoquinoline derivatives were
designed, synthesized, and evaluated for their in vitro antitumor
activity in cellular proliferation assay. We also identified them as
novel Top I inhibitors by Top I Inhibition Assay. In our research, we
had found seven compounds more potent than HCPT and TPT by
in vitro antitumor activity and as potent as CPT in inhibitory activi-
ties on Top I. For instance, compounds 9a and 10a were found to be
the most potent among all the synthesized compounds, with IC50
(
human hepatocellular liver carcinoma), A549 (nonsmall cell lung
carcinoma) and HCT-116 (human colon cancer cell line) with HCPT
and TPT as positive control. We plated 6 * 10 /ml to 8 * 10 /ml cells
per well in 96-well plates and incubated for 24 h. Over a range of
concentrations from 0.01 to 100 lM, all compounds were added
and treated for 48 h. Twenty microliters of MTT solution were
4
4
added to each well, and plates were incubated for 4 h. DMSO
(
150
in shaking table to dissolve the Formazan crystals. The absorbance
at tested wavelength of 570 nm) was measured on an
ll) was added to each well. Then shake the plate ten minutes
(
enzyme-linked immunosorbent detector. Then the cytotoxicity
O
O
O
O
O
O
H
H
3
CO
values of 0.08, 0.12
lM (HepG2), 0.048, 0.062 lM (A549) and
H
3
CO
CO
N
O
0
.117, 0.233 M (HCT-116), respectively; approximately 2–100-
l
N
N
N
N
H
3
3
CO
fold more potent than HCPT and TPT in this biological assay. Com-
pounds 9a and 10a was also as potentas CPT in Top I inhibitionassay.
Further research about the biological activities and antitumor mech-
anism of these potent compounds are currently going on in our
laboratory.
O
O
indotecan (LMP 400)
indimitecan (LMP 776)
Figure 3. Structure of indotecan (LMP 400) and indimitecan (LMP 776).